Investigation of the apical progenitors diversity

Load libraries and QCFiltered dataset

# Load library
library(Seurat)
library(monocle)
library(princurve)
library(cluster)
library(parallel)
library(ggplot2)
library(ggExtra)
library(dplyr)
library(reshape)
library(gridExtra)
library(RColorBrewer)
library(wesanderson)
library(viridis)

#Set ggplot theme as classic
theme_set(theme_classic())

# Load the full annotated dataset
Allcells.data <- readRDS("./QC.filtered.cells.RDS")

Extract Apical progenitors and compute Pseudo-DorsoVentral score

Extract apical progenitors and filter the gene expression matrix

We perform Kmeans clustering on the 2 cell state scores :

• Apical progenitors AP
• Basal progenitors BP

set.seed(100)
#K-means clustering based on AP, BP scores across cells
cl <- kmeans(cbind(Allcells.data@meta.data$AP_signature1, Allcells.data@meta.data$BP_signature1), 3)
Allcells.data@meta.data$kmeanClust <- paste0("Clust.",cl$cluster)

col.pal <- wes_palette("GrandBudapest1", 3, type = "discrete")

p1 <- ggplot(Allcells.data@meta.data, aes(x=AP_signature1, y=BP_signature1, colour = kmeanClust)) +
  scale_color_manual(values=col.pal) +
  geom_point() + 
  theme(legend.position="none")
ggMarginal(p1, type = "histogram", fill="lightgrey") ; rm(p1)

DimPlot(Allcells.data,
        group.by = "kmeanClust",
        reduction.use = "spring",
        cols.use = col.pal,
        dim.1 = 1,
        dim.2 = 2,
        do.label=T,
        label.size = 4,
        no.legend = F)

We then extract the glutamatergic neuron branche as beeing the Kmeans cluster with the highest mean Apical progenitor signature

#Fin cluster wiht the highest mean APscore
MeanKclust.APscore <- aggregate(AP_signature1 ~ kmeanClust, Allcells.data@meta.data, mean)
APclust <- MeanKclust.APscore %>% filter(AP_signature1 == max(AP_signature1)) %>% pull(kmeanClust)

#Extract apical progenitors 
barcodes <- Allcells.data@meta.data %>% filter(kmeanClust == APclust) %>% pull(Barcodes)
AP.data <-  SubsetData(Allcells.data, cells.use = barcodes , subset.raw = T,  do.clean = F)

#Further filter the 3 outlier cells based on spring coordinates
cells <- rownames(AP.data@dr$spring@cell.embeddings[AP.data@dr$spring@cell.embeddings[,2] > 250,])
AP.data <-  SubsetData(AP.data, cells.use = cells , subset.raw = T,  do.clean = F)

DimPlot(AP.data,
        group.by = "kmeanClust",
        reduction.use = "spring",
        cols.use = col.pal,
        dim.1 = 1,
        dim.2 = 2,
        do.label=T,
        label.size = 4,
        no.legend = F)

Filter gene counts matrix

#Remove non epressed genes
num.cells <- Matrix::rowSums(AP.data@data > 0)
genes.use <- names(x = num.cells[which(x = num.cells >= 20)])
AP.data@raw.data <- AP.data@raw.data[genes.use, ]
AP.data@data <- AP.data@data[genes.use, ]

#Normalize and Scale the data
AP.data <- NormalizeData(object = AP.data,
                         normalization.method = "LogNormalize", 
                         scale.factor = round(median(AP.data@meta.data$nUMI)),
                         display.progress = F)

AP.data <- FindVariableGenes(object = AP.data,
                                  mean.function = ExpMean,
                                  dispersion.function = LogVMR,
                                  x.low.cutoff = 0.0125,
                                  x.high.cutoff = 3,
                                  y.cutoff = 1, do.plot = F,
                                  display.progress = F)

AP.data <- ScaleData(object = AP.data, vars.to.regress = c("CC.Difference","percent.mito", "nUMI"), display.progress = F)

Import Spring dimensionality reduction

Cell cycle associated genes were excluded for PCA dimensionality reduction and Spring plot was generating with these parameter :

Number of cells: 1648
Number of genes that passed filter: 857
Min expressing cells (gene filtering): 3
Min number of UMIs (gene filtering): 3
Gene variability %ile (gene filtering): 90
Number of principal components: 7
Number of nearest neighbors: 20
Number of force layout iterations: 500

#Import Spring coordinates calculated without cell cycle genes
Coordinates <- read.table("./Progenitors/E12.AP.Coordinates.txt", sep=",", header = F)[,c(2,3)]
rownames(Coordinates) <- rownames(AP.data@meta.data)
AP.data <- SetDimReduction(AP.data,
                           reduction.type = "spring.AP",
                           slot = "cell.embeddings",
                           new.data = as.matrix(Coordinates))
AP.data@dr$spring.AP@key <- "spring.AP"
colnames(AP.data@dr$spring.AP@cell.embeddings) <- paste0(GetDimReduction(object = AP.data, reduction.type = "spring.AP",slot = "key"), c(1,2))

Fit a principal curve over the AP in the Spring space

data <- data.frame(springAP.1 = AP.data@dr$spring.AP@cell.embeddings[,1],
                   springAP.2 = AP.data@dr$spring.AP@cell.embeddings[,2],
                   spring1 = AP.data@dr$spring@cell.embeddings[,1],
                   spring2 = AP.data@dr$spring@cell.embeddings[,2])

# Fit the principal curve
fit <- principal_curve(as.matrix(data[,1:2]),
                       smoother='lowess',
                       trace=TRUE,
                       f = 0.7,
                       stretch=0,
                       plot_iterations = F)

## Starting curve---distance^2: 2022449778
## Iteration 1---distance^2: 745172
## Iteration 2---distance^2: 696270.5
## Iteration 3---distance^2: 677509.1
## Iteration 4---distance^2: 668251.7
## Iteration 5---distance^2: 663393.6
## Iteration 6---distance^2: 660928.6
## Iteration 7---distance^2: 659586.4
## Iteration 8---distance^2: 658917.8
## Iteration 9---distance^2: 658638.8

DorsoVentral.Score <- fit$lambda/max(fit$lambda) #The actual speudotime
pc.line <- as.data.frame(fit$s[order(fit$lambda),]) #The principal curve smoothed

data$Phase <- as.character(AP.data@meta.data$Phase)
data$DorsoVentral.Score <- DorsoVentral.Score

# Direction of the maturation score using Zbtb20 expression (reverte if positive correlation)
if (cor(data$DorsoVentral.Score, AP.data@data['Zbtb20', ]) > 0) { data$DorsoVentral.Score <- -(data$DorsoVentral.Score - max(data$DorsoVentral.Score))}

AP.data@meta.data$DorsoVentral.Score <- data$DorsoVentral.Score

#Plot Cell onto PC1 and PC2 with principal curve
ggplot(data, aes(springAP.1, springAP.2)) + 
  geom_point(aes(color=Phase), size=2, shape=16, values=col.pal) +
  geom_line(data=pc.line, color='red', size=0.77)

#Plot Speudotime color gradient on the cell cycle filtered Spring embbeding
ggplot(data, aes(springAP.1, springAP.2)) +
  geom_point(aes(color=DorsoVentral.Score), size=2, shape=16) +
  scale_color_viridis(direction = -1) +
  geom_line(data=pc.line, color='red', size=0.77)

Manuscript Fig. 5A

#Plot Speudotime color gradient on the Spring embbeding calculated from full dataset
ggplot(data, aes(spring1, spring2)) + 
  geom_point(aes(color=DorsoVentral.Score), size=2, shape=16) +
  scale_color_viridis(direction = -1) 

Find differentially expressed genes along the pseudo DV axis

Initialize a monocle object

# Transfert metadata 
meta.data <- data.frame(barcode = rownames(AP.data@meta.data),
                        Cluster = AP.data@meta.data$old.ident,
                        DorsoVentral.Score =  AP.data@meta.data$DorsoVentral.Score,
                        CellcyclePhase = AP.data@meta.data$Phase,
                        row.names = rownames(AP.data@meta.data))
                   
Annot.data  <- new('AnnotatedDataFrame', data = meta.data)

# Transfert count data
count.data = data.frame(gene_short_name = rownames(AP.data@raw.data),
                  row.names = rownames(AP.data@raw.data))

feature.data <- new('AnnotatedDataFrame', data = count.data)

# Create the CellDataSet object
gbm_cds <- newCellDataSet(as.matrix(AP.data@raw.data),
                          phenoData = Annot.data,
                          featureData = feature.data,
                          lowerDetectionLimit = 1,
                          expressionFamily = negbinomial())

gbm_cds <- estimateSizeFactors(gbm_cds)
gbm_cds <- estimateDispersions(gbm_cds)
gbm_cds <- detectGenes(gbm_cds, min_expr = 0.1)

rm(list = ls()[!ls() %in% c("AP.data", "gbm_cds")])

Test each gene trend over pseudo-DV score

# Exclude cell cycle associated genes
CCgenes <- as.character(read.table("./Progenitors/CellCycleGenes.csv", sep = "\t", header = F)[,1])
Input.genes <- AP.data@var.genes[!AP.data@var.genes %in% CCgenes]

# Perform the test for differential expression as a function of pseudo-DV score while controling for cell cycle phase
DV.Axis.genes <- differentialGeneTest(gbm_cds[Input.genes,], 
                                      fullModelFormulaStr = "~sm.ns(DorsoVentral.Score, df = 3)*CellcyclePhase", 
                                      reducedModelFormulaStr = "~CellcyclePhase", 
                                      cores = detectCores() -2)

# Filter genes with a FDR < 0.001
DV.Axis.genes.FDR.filtered <- DV.Axis.genes %>% filter(qval < 1e-3)

Smooth significative gene expression pseudo-DV axis

# Create a new pseudo-DV vector of 500 points
nPoints <- 500
new_data <- data.frame(DorsoVentral.Score = seq(min(pData(gbm_cds)$DorsoVentral.Score), max(pData(gbm_cds)$DorsoVentral.Score), length.out = nPoints))

# Smooth gene expression
Smooth.curve.matrix <- genSmoothCurves(gbm_cds[as.character(DV.Axis.genes.FDR.filtered$gene_short_name),],
                                       trend_formula = "~sm.ns(DorsoVentral.Score, df = 3)",
                                       relative_expr = TRUE,
                                       new_data = new_data,
                                       cores= detectCores() - 2)

Cluster gene by similar profiles over pseudo-DV axis

set.seed(100)
# Cluster genes using the Partitioning Around Medoids algorithm
DV.Axis.genes.clusters <- pam(as.dist((1-cor(Matrix::t(Smooth.curve.matrix), method = "spearman"))), k=9)
table(DV.Axis.genes.clusters$clustering)

## 
##   1   2   3   4   5   6   7   8   9 
## 122  82 104  42  50  45  21  23  48

# Store the results 
Gene.dynamique <- data.frame(Gene= names(DV.Axis.genes.clusters$clustering),
                             pval= DV.Axis.genes.FDR.filtered$pval,
                             qval=DV.Axis.genes.FDR.filtered$qval,
                             num_cells_expressed=DV.Axis.genes.FDR.filtered$num_cells_expressed,
                             Waves = DV.Axis.genes.clusters$clustering) %>% arrange(Waves)

row.names(Gene.dynamique) <- Gene.dynamique$Gene
Gene.dynamique$Gene.Clusters <- paste0("Clust.",Gene.dynamique$Waves)

write.table(Gene.dynamique, "./Progenitors/Gene.dynamique.csv", sep = ";", quote = F)

# Load custom plotting functions
source("./functions/GenesTrendPlots.R")

#Plot gene clusters trends
Clusters.trend(AP.data,
               Which.cluster = 1:9,
               clust.list = DV.Axis.genes.clusters,
               group.by = "global",
               span = 1,
               Smooth.method = "auto",
               Use.scale.data = T)

Manuscript Fig. 5C

Cluster cell by similar expression profiles on the pseudo-DV axis

set.seed(100)
# Cluster cells using the Partitioning Around Medoids algorithm
Cells.Clust <- pam(as.dist((1 - cor(Smooth.curve.matrix ,method = "spearman"))), k=7)
Domaines.Clust <- data.frame(Domaines = paste0("Clust.",Cells.Clust$clustering))

Plot gene/cell heatmap

# Re-order gene expression cluster along the axis
Sorted.gene.dyn <- Gene.dynamique %>% arrange(factor(Gene.Clusters, levels = paste0("Clust.",c(7,2,6,3,1,9,5,4,8))))
rownames(Sorted.gene.dyn) <- Sorted.gene.dyn$Gene

anno.colors <- list(Domaines = c(Clust.1="#83c3b8", Clust.2="#009fda", Clust.3="#3e69ac", Clust.4="#e46b6b", Clust.5="#e3c148", Clust.6="#b7d174", Clust.7="#68b041"),
                    Gene.Clusters = c(Clust.1 ="#cc3a1b" , Clust.2="#046c9a", Clust.3="#e7823a", Clust.4="#cc8778", Clust.5="#68b041", Clust.6="#5ab793", Clust.7="#e3c148", Clust.8="#e46B6b", Clust.9="#b79f0b"))

pheatmap::pheatmap(Smooth.curve.matrix[as.character(Sorted.gene.dyn$Gene),],
                   scale = "row",
                   cluster_rows = F,
                   cluster_cols = F,
                   gaps_col = cumsum(as.numeric(table(Domaines.Clust$Domaines))),
                   gaps_row = cumsum(as.numeric(table(Sorted.gene.dyn$Gene.Clusters)[paste0("Clust.",c(7,2,6,3,1,9,5,4,8))])) ,
                   annotation_row = Sorted.gene.dyn %>% dplyr::select(Gene.Clusters),
                   annotation_col = Domaines.Clust,
                   annotation_colors = anno.colors,
                   show_colnames = F,
                   show_rownames = T,
                   fontsize_row = 2,
                   color = rev(brewer.pal(11,"RdBu")),
                   breaks = seq(-2.5,2.5, length.out = 11),
                   main = "Genes expression along Dorso-Ventral axis")

Manuscript Fig. 5C

Assign domain identity

We assign domain identity based on clusters’ transcriptional profile by setting boundaries over pseudo-dv score

# Set the boundary over speudotime score
new_data$cluster <- Domaines.Clust$Domaines
Infered.Domain.boundary <- aggregate(DorsoVentral.Score ~ cluster, new_data, max) %>% pull(DorsoVentral.Score)

# Assign identity based on the position of the cell on the pseudo-dv axis
Domaine.Ident <- sapply(AP.data@meta.data$DorsoVentral.Score,
                        function(x){ if(x<Infered.Domain.boundary[1]){ x = "Sub.Pallium.1"
                        } else if(x> Infered.Domain.boundary[1] & x< Infered.Domain.boundary[2]){ x ="Sub.Pallium.2"
                        } else if(x> Infered.Domain.boundary[2] & x< Infered.Domain.boundary[3]){ x = "Sub.Pallium.3" 
                        } else if(x> Infered.Domain.boundary[3] & x< Infered.Domain.boundary[4]){ x = "Ventral.Pallium"
                        } else if(x> Infered.Domain.boundary[4] & x< Infered.Domain.boundary[5]){ x = "lateral.Pallium.1"
                        } else if(x> Infered.Domain.boundary[6]){ x = "Dorsal.Pallium"
                        } else x="lateral.Pallium.2"})

# Transfert the identity to the Seurat object
AP.data@meta.data$Domaine <- Domaine.Ident
AP.data <- SetAllIdent(AP.data, id = "Domaine")

Plot.Genes.trend(AP.data,
                 genes = c("Gsx2", "Dbx1", "Gm29260", "Tfap2c", "Emx1", "Lrrn1"),
                 Use.scale.data = F)

Manuscript Fig. 5D

DimPlot(AP.data,
         group.by = "Domaine",
         reduction.use = "spring",
         dim.1 = 1,
         dim.2 = 2,
         do.label=F,
         label.size = 4,
         no.legend = F,
         cols.use = tolower(c("#68B041", "#E3C148", "#B7D174", "#83C3B8", "#009FDA", "#3E69AC", "#E46B6B"))
         )

Plot Figure 5B

#load full dataset
Allcells.data <- readRDS("./QC.filtered.cells.RDS")

#Transfer the identities
Rename.Clust <-  function(Clustdata, RawQCdata) {
  unClustered.cells <- RawQCdata@meta.data$Barcodes
  RawQCdata <- SetIdent(RawQCdata, cells.use = unClustered.cells, ident.use = "All.Unclustered.Cells")
  
  for(i in unique(Clustdata@meta.data$Domaine)){
  New.ident <- i
  Barcodes <- rownames(subset(Clustdata@meta.data, Clustdata@meta.data$Domaine == i))
  print(paste0("Cluster_",i,": ",length(Barcodes), " Cells"))
  Barcodes <- Barcodes[Barcodes %in% rownames(RawQCdata@meta.data)]
  RawQCdata <- SetIdent(RawQCdata, cells.use = Barcodes ,ident.use = paste0("AP.",i))
  }
  return(RawQCdata)
}

Allcells.data <- Rename.Clust(Clustdata = AP.data, RawQCdata = Allcells.data)

## [1] "Cluster_Sub.Pallium.2: 363 Cells"
## [1] "Cluster_Ventral.Pallium: 312 Cells"
## [1] "Cluster_Sub.Pallium.3: 197 Cells"
## [1] "Cluster_lateral.Pallium.1: 219 Cells"
## [1] "Cluster_Dorsal.Pallium: 201 Cells"
## [1] "Cluster_lateral.Pallium.2: 113 Cells"
## [1] "Cluster_Sub.Pallium.1: 241 Cells"

colors <-  c("#969696",tolower(c("#68B041", "#E3C148", "#B7D174", "#83C3B8", "#009FDA", "#3E69AC", "#E46B6B")))

DimPlot(Allcells.data,
        reduction.use = "spring", 
        dim.1 = 1,
        dim.2 = 2,
        do.label=T,
        label.size = 2,
        no.legend = T,
        cols.use = colors)

Manuscript Fig. 5B

rm(list = ls()[!ls() %in% c("AP.data")])

Plot the representative gene of the Figure S6

# Load custom plotting functions
source("./functions/GenesTrendPlots.R")

Cluster 6

Plot.Genes.trend(AP.data,
                 genes = c("Dlx1", "Ano1", "Dlx2", "Olig2"),
                 Use.scale.data = F)

Manuscript Fig.S6A

Cluster 2

Plot.Genes.trend(AP.data,
                 genes = c("Otx2", "Zbtb20", "Six3", "Ascl1"),
                 Use.scale.data = F)

Manuscript Fig.S6B

Cluster 3

Plot.Genes.trend(AP.data,
                 genes = c("Meis1", "Rorb", "Epha3"),
                 Use.scale.data = F)

Manuscript Fig.S6C

Cluster 1

Plot.Genes.trend(AP.data,
                 genes = c("Sfrp2", "Eva1c", "Sema5a", "Fat4"),
                 Use.scale.data = F)

Manuscript Fig.S6D

Cluster 9

Plot.Genes.trend(AP.data,
                 genes = c("Dmrta2","Lypd6", "Pax6", "Dmrta1"),
                 Use.scale.data = F)

Manuscript Fig.S6E

Cluster 5 ?

Plot.Genes.trend(AP.data,
                 genes = c("Emx1", "Arx", "Lmo3"),
                 Use.scale.data = F)

Manuscript Fig.S6F

Cluster 9

Plot.Genes.trend(AP.data,
                 genes = c("Fezf2", "Emx2", "Sp8"),
                 Use.scale.data = F)

Manuscript Fig.S6G

Transfert these ident on the full dataset (Manuscript Fig. 2A)

#load full dataset
Allcells.data <- readRDS("./Clustered.cells.RDS")

#Transfer the identities
Rename.Clust <-  function(Clustdata, RawQCdata) {
  for(i in unique(Clustdata@meta.data$Domaine)){
    New.ident <- i
    Barcodes <- rownames(subset(Clustdata@meta.data, Clustdata@meta.data$Domaine == i))
    print(paste0("Cluster_",i,": ",length(Barcodes), " Cells"))
    Barcodes <- Barcodes[Barcodes %in% rownames(RawQCdata@meta.data)]
    RawQCdata <- SetIdent(RawQCdata, cells.use = Barcodes ,ident.use = paste0("AP.",i))
  }
  return(RawQCdata)
}

Allcells.data <- Rename.Clust(Clustdata = AP.data, RawQCdata = Allcells.data)

## [1] "Cluster_Sub.Pallium.2: 363 Cells"
## [1] "Cluster_Ventral.Pallium: 312 Cells"
## [1] "Cluster_Sub.Pallium.3: 197 Cells"
## [1] "Cluster_lateral.Pallium.1: 219 Cells"
## [1] "Cluster_Dorsal.Pallium: 201 Cells"
## [1] "Cluster_lateral.Pallium.2: 113 Cells"
## [1] "Cluster_Sub.Pallium.1: 241 Cells"

colors2 <-  c("#969696",
              tolower(c("#68B041", "#E3C148", "#B7D174", "#83C3B8", "#009FDA", "#3E69AC", "#E46B6B")),
              "#ec756d", "#c773a7", "#7293c8", "#b79f0b", "#3ca73f","#31b6bd",
              "#ebcb2e", "#9ec22f", "#a9961b", "#cc3a1b", "#cc8778" , "#d14c8d", "#4cabdc", "#5ab793", "#e7823a","#e6bb9b", "#046c9a", "#4784a2" , "#4990c9")

DimPlot(Allcells.data,
        reduction.use = "spring", 
        dim.1 = 1,
        dim.2 = 2,
        do.label=T,
        label.size = 2,
        no.legend = T,
        cols.use = colors2)

Manuscript Fig. 2A

Allcells.data <- StashIdent(Allcells.data, save.name = "Cluster.ident")
saveRDS(Allcells.data, "./Clustered.cells.RDS")
saveRDS(AP.data, "./AP.data.RDS")

Session Info

#date
format(Sys.time(), "%d %B, %Y, %H,%M")

## [1] "05 novembre, 2020, 11,24"

#Packages used
sessionInfo()

## R version 3.6.3 (2020-02-29)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 18.04.5 LTS
## 
## Matrix products: default
## BLAS:   /usr/lib/x86_64-linux-gnu/atlas/libblas.so.3.10.3
## LAPACK: /usr/lib/x86_64-linux-gnu/atlas/liblapack.so.3.10.3
## 
## locale:
##  [1] LC_CTYPE=fr_FR.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=fr_FR.UTF-8        LC_COLLATE=fr_FR.UTF-8    
##  [5] LC_MONETARY=fr_FR.UTF-8    LC_MESSAGES=fr_FR.UTF-8   
##  [7] LC_PAPER=fr_FR.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=fr_FR.UTF-8 LC_IDENTIFICATION=C       
## 
## attached base packages:
##  [1] splines   stats4    parallel  stats     graphics  grDevices utils    
##  [8] datasets  methods   base     
## 
## other attached packages:
##  [1] viridis_0.5.1       viridisLite_0.3.0   wesanderson_0.3.6  
##  [4] RColorBrewer_1.1-2  gridExtra_2.3       reshape_0.8.8      
##  [7] dplyr_0.8.3         ggExtra_0.9         cluster_2.1.0      
## [10] princurve_2.1.4     monocle_2.14.0      DDRTree_0.1.5      
## [13] irlba_2.3.3         VGAM_1.1-2          Biobase_2.46.0     
## [16] BiocGenerics_0.32.0 Seurat_2.3.4        Matrix_1.2-17      
## [19] cowplot_1.0.0       ggplot2_3.2.1      
## 
## loaded via a namespace (and not attached):
##   [1] snow_0.4-3           backports_1.1.5      Hmisc_4.3-0         
##   [4] plyr_1.8.4           igraph_1.2.5         lazyeval_0.2.2      
##   [7] densityClust_0.3     fastICA_1.2-2        digest_0.6.25       
##  [10] foreach_1.4.7        htmltools_0.5.0      lars_1.2            
##  [13] gdata_2.18.0         magrittr_1.5         checkmate_1.9.4     
##  [16] mixtools_1.1.0       ROCR_1.0-7           limma_3.42.0        
##  [19] matrixStats_0.55.0   R.utils_2.9.0        docopt_0.6.1        
##  [22] colorspace_1.4-1     ggrepel_0.8.1        xfun_0.18           
##  [25] sparsesvd_0.2        crayon_1.3.4         jsonlite_1.7.0      
##  [28] zeallot_0.1.0        survival_2.44-1.1    zoo_1.8-6           
##  [31] iterators_1.0.12     ape_5.3              glue_1.4.1          
##  [34] gtable_0.3.0         kernlab_0.9-29       prabclus_2.3-1      
##  [37] DEoptimR_1.0-8       scales_1.1.0         pheatmap_1.0.12     
##  [40] bibtex_0.4.2         miniUI_0.1.1.1       Rcpp_1.0.5          
##  [43] metap_1.1            dtw_1.21-3           xtable_1.8-4        
##  [46] htmlTable_1.13.2     reticulate_1.13      foreign_0.8-72      
##  [49] bit_4.0.4            proxy_0.4-23         mclust_5.4.5        
##  [52] SDMTools_1.1-221.1   Formula_1.2-3        tsne_0.1-3          
##  [55] htmlwidgets_1.5.1    httr_1.4.1           FNN_1.1.3           
##  [58] gplots_3.0.1.1       fpc_2.2-3            acepack_1.4.1       
##  [61] modeltools_0.2-22    ica_1.0-2            farver_2.0.1        
##  [64] pkgconfig_2.0.3      R.methodsS3_1.7.1    flexmix_2.3-15      
##  [67] nnet_7.3-14          labeling_0.3         tidyselect_0.2.5    
##  [70] rlang_0.4.7          reshape2_1.4.3       later_1.0.0         
##  [73] munsell_0.5.0        tools_3.6.3          ggridges_0.5.1      
##  [76] fastmap_1.0.1        evaluate_0.14        stringr_1.4.0       
##  [79] yaml_2.2.1           npsurv_0.4-0         knitr_1.26          
##  [82] bit64_4.0.2          fitdistrplus_1.0-14  robustbase_0.93-5   
##  [85] caTools_1.17.1.2     purrr_0.3.3          RANN_2.6.1          
##  [88] pbapply_1.4-2        nlme_3.1-141         mime_0.7            
##  [91] slam_0.1-46          R.oo_1.23.0          hdf5r_1.3.2.9000    
##  [94] compiler_3.6.3       rstudioapi_0.11      png_0.1-7           
##  [97] lsei_1.2-0           tibble_2.1.3         stringi_1.4.6       
## [100] highr_0.8            lattice_0.20-41      HSMMSingleCell_1.6.0
## [103] vctrs_0.2.0          pillar_1.4.2         lifecycle_0.1.0     
## [106] combinat_0.0-8       Rdpack_0.11-0        lmtest_0.9-37       
## [109] data.table_1.12.6    bitops_1.0-6         gbRd_0.4-11         
## [112] httpuv_1.5.2         R6_2.4.1             latticeExtra_0.6-28 
## [115] promises_1.1.0       KernSmooth_2.23-15   codetools_0.2-16    
## [118] MASS_7.3-53          gtools_3.8.1         assertthat_0.2.1    
## [121] withr_2.1.2          qlcMatrix_0.9.7      mgcv_1.8-33         
## [124] diptest_0.75-7       doSNOW_1.0.18        grid_3.6.3          
## [127] rpart_4.1-15         tidyr_1.0.0          class_7.3-17        
## [130] rmarkdown_2.5        segmented_1.0-0      Rtsne_0.15          
## [133] shiny_1.4.0          base64enc_0.1-3

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1. Institute of Psychiatry and Neuroscience of Paris, INSERM U1266, 75014, Paris, France↩